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Hi, guys. I'm trying to construct a PE500 library from 50ng genomic DNA by using the Nextera transposase. However, the fragment size always concentrate on 200bp even I have chosen the HMW buffer. How can I control the MW of the fragments? Or is there anyone have similar experiences?
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Hi Kitty,
Not sure why you started another thread rather than continuing your old one...
From your bioanalzyer plot:
It looks like most of your library is ending up in annealed concatamers. How about denaturing it (2 minutes 95 oC, followed immediately by chilling on ice) and then running on an pico RNA bioanalyzer chip?
But if that is not possible, you could try using way less cycles of amplification but then use a DNA High Sensitivity Agilient Chip. That way you can see your library products without needing to do so many PCR cycles that your primers run low and you get high levels of amplicon daisy chaining (or bird nesting, or whatever you want to call it.)
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Phillip -
nextera for targeted exon capture?
is nextera library prep OK to do exon capture?Comment
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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